Testosterone Related to Age and Life-History Stages in Male Baboons and Geladas

ARTICLE IN PRESS YHBEH-02887; No. of pages: 9; 4C: Hormones and Behavior xxx (2009) xxx–xxx

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Hormones and Behavior

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Testosterone related to age and life-history stages in male baboons and geladas

Jacinta C. Beehner a,b,⁎,1, Laurence Gesquiere c,1, Robert M. Seyfarth d, Dorothy L. Cheney e, Susan C. Alberts f,g, Jeanne Altmann c,g,h a Department of Psychology, University of Michigan, Ann Arbor, MI 48109, USA b Department of Anthropology, University of Michigan, Ann Arbor, MI 48109, USA c Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 08544, USA d Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104, USA e Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA f Department of Biology, Duke University, Durham, NC 27708, USA g Institute of Primate Research, National Museums of Kenya, Nairobi, Kenya h Department of Animal Physiology, University of Nairobi, Nairobi, Kenya article info abstract

Article history: Despite significant advances in our knowledge of how testosterone mediates life-history trade-offs, this Received 26 April 2009 research has primarily focused on seasonal taxa. We know comparatively little about the relationship Revised 11 August 2009 between testosterone and life-history stages for non-seasonally breeding species. Here we examine Accepted 12 August 2009 testosterone profiles across the life span of males from three non-seasonally breeding primates: yellow Available online xxxx baboons (Papio cynocephalus or P. hamadryas cynocephalus), chacma baboons (Papio ursinus or P. h. ursinus), and geladas (Theropithecus gelada). First, we predict that testosterone profiles will track the reproductive Keywords: fi Androgen pro les of each taxon across their respective breeding years. Second, we evaluate age-related changes in Fecal steroid testosterone to determine whether several life-history transitions are associated with these changes. Hormone Subjects include males (N2.5 years) from wild populations of each taxon from whom we had fecal samples Life history for hormone determination. Although testosterone profiles across taxa were broadly similar, considerable Maturation variability was found in the timing of two major changes: (1) the attainment of adult levels of testosterone Method validation and (2) the decline in testosterone after the period of maximum production. Attainment of adult testosterone levels was delayed by 1 year in chacmas compared with yellows and geladas. With respect to the decline in testosterone, geladas and chacmas exhibited a significant drop after 3 years of maximum production, while yellows declined so gradually that no significant annual drop was ever detected. For both yellows and chacmas, increases in testosterone production preceded elevations in social dominance rank. We discuss these differences in the context of ecological and behavioral differences exhibited by these taxa. © 2009 Elsevier Inc. All rights reserved.

Introduction that high T facilitates inter-male competition at times in the life cycle when males need to compete for receptive females or the resources The steroid hormone testosterone (T) is known to affect many necessary to attract such females. However, because high levels of T vertebrate life-history traits and has been implicated as a mediator of may interfere with paternal behavior (e.g., Goymann et al., 2007; Gray life-history trade-offs (Hau, 2007; Ricklefs and Wikelski, 2002; et al., 2006; Muller et al., 2009; Nunes et al., 2000, 2001), T levels reviewed in Zera and Harshman, 2001). For example, the increase in should decrease when males care for offspring. Formulation of the production of T when males reach puberty and begin to seek out challenge hypothesis was based on monogamous, seasonal birds with mating opportunities is at the same time associated with costs, such as a high degree of paternal care and cycles of mating and care within reduced immune function (McGlothlin et al., 2007). each year. For such seasonal species with relatively short and intense One model for T-behavior trade-offs, known as the “challenge cycles of mating, the period of interest for investigating T-mediated hypothesis”, proposes that variation in male T across life-history trade-offs is within each breeding season (temperate birds: e.g., stages reflects differential allocation to mating and parenting behavior McGlothlin et al., 2007; Wingfield et al., 1990; tropical birds: e.g., Hau (Wingfield et al., 1990). Specifically, the challenge hypothesis predicts et al., 2008; reptiles: e.g., Wack et al., 2008; and mammals: e.g., Brockman et al., 2001; Cavigelli and Pereira, 2000; Malo et al., 2009; Moss et al., 2001; Ostner et al., 2002, 2008). ⁎ Corresponding author. Department of Psychology, University of Michigan, 530 More recently, the hypothesis has been modified to apply to non- Church St., Ann Arbor, MI 48109-1043, USA. Fax: +1 734 763 7480. E-mail address: [email protected] (J.C. Beehner). seasonally breeding species (Archer, 2006; Muller and Wrangham, 1 Authors Beehner and Gesquiere contributed equally toward this manuscript. 2004). For non-seasonal species, T changes across the year are less

Please cite this article as: Beehner, J.C., et al., Testosterone related to age and life-history stages in male baboons and geladas, Horm. Behav. (2009), doi:10.1016/j.yhbeh.2009.08.005 ARTICLE IN PRESS

2 J.C. Beehner et al. / Hormones and Behavior xxx (2009) xxx–xxx informative than T changes across the life span (e.g., Bribiescas, 2006; Although high-dominance rank facilitates reproductive access Crawford et al., 1997; Ellison et al., 2002; Martin et al., 1977). to females in both baboon taxa, behavioral data and paternity Therefore, with respect to T-mediated trade-offs for non-seasonal determination indicate that non-alpha males are thought to be species, T should be up-regulated at the start of reproductive more viable competitors for females in yellow baboon groups than maturity, maintained throughout the breeding years, and down- they are in chacma groups (Alberts et al., 2003, 2006; Bulger, 1993; regulated once males no longer breed or when they focus on paternal Cheney and Seyfarth, unpublished data). Mating in some chacma behaviors. For non-seasonal species, many of the potential trade-offs populations may therefore at any one time be relatively more extend across life-history stages that can take years for long-lived unimale than yellow baboons due to higher reproductive skew. organisms. With the exception of several studies of human and Furthermore, the timing of paternal care for chacma males may captive non-human primates (e.g., Bribiescas, 2001, 2005, 2006; also parallel that described for geladas because (1) paternal care Crawford et al., 1997; Ellison et al., 2002; Martin et al., 1977), serves to protect offspring from infanticide (Palombit et al., 2000), investigation of T profiles across the entire life span is rare (but see (2) infanticidal males are generally newly immigrant males that chimpanzees (Pan troglodytes): Seraphin et al., 2008; and mandrills have attained the alpha position in the dominance hierarchy (Bulger (Mandrillus sphinx): Setchell and Dixson, 2002). and Hamilton, 1987; Busse and Hamilton, 1981; Collins et al., 1984; Here we examine T profiles across the life span of males in wild Palombit et al., 2000; Tarara, 1987), and therefore (3) paternal populations of three long-lived, non-seasonally breeding primate behavior generally occurs after a father has fallen from the alpha taxa (see Methods section): yellow baboons (Papio cynocephalus position. or P. hamadryas cynocephalus), chacma baboons (Papio ursinus or Based on these differences, two predictions emerge. If, as in P. h. ursinus), and geladas (Theropithecus gelada). Specifically, we many bird species (Wingfield et al., 1990), T profiles are linked evaluate age-related changes in T and examine whether several primarily to demographically defined mating systems, gelada T life-history transitions (or “maturational milestones”) are associ- profiles will exhibit a more discrete period of elevation while those ated with these changes. Despite the rarity of mammalian paternal of yellow and chacma baboons will exhibit a more extended period care, baboon and gelada males are known to invest in some degree of adult T levels. Alternatively, if T profiles track the actual differ- of paternal care (Beehner and Bergman, 2008; Buchan et al., 2003; ences among taxa in reproductive access to females and parenting Dunbar, 1984; Moscovice et al., 2009; Palombit et al., 2000). behavior, geladas and chacmas will exhibit a discrete period of T Therefore, we expect that trade-offs between the high levels of T elevation, and yellow males will exhibit a more extended period of optimal for mating and the low levels of T optimal for parenting adult T levels, characterized by a gradual fall in T after peak repro- will result in T modulation for these three taxa, and that this ductive years. modulation will reflect differences among them in their respective Third, we describe the relationship between the maturational rise life histories. in T levels and several male maturational milestones. One visible We have three lines of inquiry. First, as a physiological validation maturational marker, (1) enlargement of testes, is available for only and in accordance with T profiles from other vertebrate species, we one taxon (yellow baboons) and has been shown to precede test the prediction that juvenile males have significantly lower fecal T significant increases in fecal T metabolites (Gesquiere et al., 2005). metabolites than adult males. Further, based on profiles of T across the Therefore, we examine the relationship in these three taxa between T human male life span, T for all three taxa should exhibit an inverse U- profiles and four additional maturational markers: (2) timing of natal shaped pattern, exhibiting a rise at or around maturity and a decline dispersal, (3) acquisition of adult dominance rank, (4) first sexual as the animals senesce. consortship, and (5) acquisition of highest rank. Second, we make the general prediction that the T profiles for males will follow the reproductive profiles of each taxon across their Methods respective breeding periods. In particular, we expect T to remain elevated during ages when males are reproductively active and to Because the taxonomic level of the different Papio groups remains return to pre-reproductive levels when mating activity declines and uncertain (Jolly, 1993), we avoid this debate altogether by referring to parental care increases. Although all three taxa are non-seasonal yellow and chacma baboons throughout as different “taxa” (see also breeders, they differ in the timing of reproductive and paternal Barrett and Henzi, 2008)—whether they are considered different behavior. At one extreme, geladas have a unimale system (polygy- species or subspecies does not affect the results presented here. nous), in which one male (“leader male”) has sole reproductive access Subjects for this study include all males aged 2.5 years and older from to females that make up a one-male unit. Gelada males have (1) a each study population from whom we had fecal samples for hormone single tenure as a leader, (2) no reproductive access to females before determination. obtaining a unit (when they live as “bachelor males” in all-male groups), and (3) no reproductive access to females after losing their Yellow baboon data collection unit (when they live as “follower males” in their former unit). For gelada males, changes in mating activity across the life span are The data for yellow baboons come from multiple groups in the qualitative and reproductive tenure is discrete. Furthermore, once Amboseli Basin, Kenya. Because individual life-history data for leader males relinquish their unit and become follower males, then members of these study groups cover more than three decades and only then do they engage in protective parenting behavior (Alberts et al., 2003, 1996; Alberts and Altmann, 1995a; Altmann and (Dunbar, 1984)—presumably to protect their offspring from infanti- Alberts, 2003; Altmann et al., 1988; Pereira, 1988; Shopland, 1987), cide (Beehner and Bergman, 2008). birthdates are known (within a few days) for all immature and many In contrast, yellow and chacma baboons exhibit a multimale social mature males. Ages of immigrant males for whom birthdates are not structure with a mating system that is polygynandrous to varying known were estimated using an established protocol based on body degrees. Male dominance rank mediates temporary reproductive size and other age-related physical characteristics when these males access to fertile females. Specifically, dominance rank functions as a first appear in one of the study groups. Timing of male maturational queue for mating opportunities (Alberts et al., 2003; Altmann, 1962; milestones for yellow baboons used in this study is taken from Bulger, 1993; Weingrill et al., 2003, 2000). For yellow and chacma previous studies on the population (see summary in Charpentier et al., males, changes in mating activity across the life span are quantitative, 2008). Male dominance ranks were determined by assigning wins and such that mating activity rises during early adulthood and falls in late losses for all dyadic agonist encounters between males, as described adulthood. in Hausfater (1975) and Alberts et al. (2003).

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As part of the continuing Amboseli baboon research, repeated fecal the solution (by inverting it 10 times), and loaded it onto a prepped, samples are collected opportunistically from all group members. solid-phase extraction cartridge (Sep-Pak Plus; Waters) followed by Because the testosterone RIA kit previously used in our laboratory 2 ml of a sodium azide solution (0.1%) as a wash and preservative. All (Equate 125I Testosterone RIA kit; SolidPhase, Portland, ME) was samples were stored dry on cartridges in separate sealed bags with discontinued, we validated a subset of our samples using a new T RIA silica beads (∼2 g) at subzero temperatures (−10 °C) until trans- kit (Diagnostics Systems Laboratories; Beckman Coulter, Webster, TX). ported to the University of Michigan for analysis. In the laboratory, For this validation, we used 2570 samples from 125 different males, for steroids were eluted from cartridges with 2.5 ml 100% methanol and an average of 21 fecal samples per male, with at least 20 different subsequently stored at −20 °C until the time of RIA. males per age category (see age categories below). All data collection procedures adhered to the Institutional Animal Care and Use Gelada data collection Committee guidelines of Princeton University and the laws of Kenya. Fecal sample collection, storage, and extraction were performed as The data for the geladas come from two bands of wild-feeding described previously (Beehner et al., 2006b; Gesquiere et al., 2005, geladas in the Simien Mountains National Park, Ethiopia. Because daily 2007; Khan et al., 2002; Lynch et al., 2003). In brief, freshly deposited observations on this gelada population began in January 2006, all ages samples were mixed thoroughly, placed in 95% ethanol, and stored in of gelada males are necessarily estimated. We placed males in age a charcoal refrigerator (∼20–25 °C) until shipped to the University of categories based on (1) a combination of published descriptions of Nairobi (every 2 weeks), where the ethanol was evaporated and the gelada age characteristics based on morphological traits (Dunbar and samples were freeze-dried. Following freeze-drying, samples were Dunbar, 1975) and (2) our observations of physical size and develop- stored at −20 °C until shipped to Princeton University. After mental markers (e.g., canine eruption) as compared to baboons (Papio) transport, each fecal sample was sifted to remove vegetative matter, of known ages. Age categories were assigned independently by two and 0.2 g of fecal powder was extracted into 2 ml 90% methanol using observers, and both sets were in close agreement (age categories, a multipulse vortexer for 30 min. Following extraction, samples were estimated ages, and general characteristics describing each category run through a prepped Oasis cartridge (Waters, Milford, MA) for can be found in Supplementary material). No overt dominance hierar- further purification. Prior to assay, all samples were stored at −20 °C. chy among gelada leader males has been reported (Mori, 1979). Juvenile males may form temporary hierarchies with their peers and Chacma baboon data collection these hierarchies might later extend to all-male groups (Dunbar, 1984); however, no dominance data are available for either of these groups. The data for the chacma baboons come from one wild-feeding Timing of first sexual consortships for gelada males was recorded as the group in the Moremi Game Reserve, Botswana. This group has been first time a new leader male mated with one of the females in his unit. studied almost continuously since 1982 (Bulger and Hamilton, 1987; Samples available for this study were derived from the first Cheney et al., 2004), and the ages of all natal males are known. The 6 months of collection. In total, we collected 328 fecal samples for ages of immigrant males were estimated based on body size and tooth hormone analysis from 100 different males, for an average of 3 wear (Kitchen et al., 2003). If newly immigrated males appeared samples per male (range: 1–10), with at least 10 individuals in each young and in their prime, they were assigned the median age at age category. Hormones were extracted from feces in the field using a dispersal for this population (9.25 years, N=26) at the time they method almost identical to that for the chacma baboons described entered the study group. Only natal emigrants who were later seen in above (with a few modifications to the volume of sample and a neighboring group were used to calculate median age at first solutions used). In brief, fresh fecal samples were thoroughly mixed, dispersal. Dominance ranks of all males were calculated monthly an aliquot of the sample (∼ 0.1 g) was placed in 3 ml of a methanol/ based on the outcomes of dyadic interactions. Males were assigned an acetone solution (4:1), and the solution was immediately homoge- adult rank after achieving dominance over another adult male. First nized. The dry weight of all fecal samples was later determined consortships were recorded after a male exhibited his first mate- (±0.001 g). Approximately 7 h later, 2.5 ml of fecal homogenate was guarding episode (see also Alberts and Altmann, 1995a). filtered through a 0.2-μm PTFE filter and washed with an additional As part of a 2-year study from 2001 to 2003, repeated fecal samples 1 ml of methanol/acetone (4:1). We then added 7 ml of distilled were collected from all adult males. Additionally, as part of a short-term water to the filtered homogenate, mixed the solution, loaded it onto a study to target all age groups, repeated fecal samples were collected prepped Sep-Pak cartridge, and washed the cartridge with 1 ml of a from males of all ages during August 2007. As for samples from the sodium azide solution (0.1%). All samples were stored dry on yellow baboons, chacma fecal samples had previously been analyzed cartridges in separate sealed bags with ∼2 g silica beads. Samples with the Equate T RIA kit. Therefore, we validated chacma fecal samples were stored at ambient temperatures for up to 10 days until they with the new DSL T RIA kit as well. For the validation, we used 726 could be transported to a freezer at a nearby lodge. Once frozen, samples from 41 different males, for an average of 18 fecal samples per samples remained at subzero temperatures (−10 °C) until trans- male. All data collection procedures adhered to the Institutional Animal ported to the University of Michigan for analysis. In the laboratory, Care and Use Committee guidelines of the University of Pennsylvania steroids were eluted from cartridges with 2.5 ml 100% methanol and and the University of Michigan and the laws of Botswana. subsequently stored at −20 °C until the time of RIA. Hormones were extracted from feces in the field using the method described by Beehner and Whitten (2004). Specifically, fresh fecal Testosterone RIA samples were mixed thoroughly, an aliquot of the sample (∼ 0.5 g) was placed in 10 ml of a methanol/acetone solution (4:1), and the For all three taxa, we used the same assay methods and the same T solution was immediately homogenized for 1 min using a battery- primary antibody. We assayed all samples for T metabolites using a powered vortexer (BioVortexer; BioSpec Products, Inc., Bartlesville, modified protocol from a commercially available T RIA kit (Diagnostics OK). The dry weight of all fecal samples was later determined Systems Laboratories; Beckman Coulter). All assays used the standards, (±0.001 g) using a battery-powered, portable scale (Ohaus Scout Pro, the primary antibody, the labeled testosterone, and the precipitant Pine Brook, NJ). Approximately 7 h later, 4.0 ml of the fecal solution provided by the kit. Working buffer was a charcoal adsorbed homogenate was filtered through a 0.2-μm polytetrafluoroethylene human serum similar to the buffer in which the standards were diluted (PTFE) syringeless filter (Whatman, Florham Park, NJ), and the filter (American Biological Technologies, Inc., Seguin, TX). The primary was subsequently washed with 1 ml of methanol/acetone (4:1). We antibody from the DSL T kit cross-reacts 100% with testosterone, 6.6% then added 7 ml of distilled water to the filtered homogenate, mixed with 5α-dihydrotestosterone, 2.2% with 5-androstane-3β,17β-diol,

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1.8% with 11-oxotestosterone, 0.9% with androstenedione, and 0.6% markers vary for each taxon in this study, we broadly define “juveniles” with 5β-dihydrotestosterone. Cross reactivity of the antiserum with all as males between the ages of 2.5 years (when males of all three taxa are other steroids is less than 0.5%. All samples were run in duplicate, and independent from their mother) and 4.5 years (when none of the three the results are expressed as ng/g dry fecal matter. taxa have reached puberty). We define adults using similar criteria; “adults” in this study comprise males older than 8.0 years (when all Method validation three taxa have reached full adulthood). Males between juveniles and adults are in various stages approaching adulthood, and these First, to evaluate the effectiveness of extracting T metabolites from differences are taxon specific. Thus, for convenience herein, we broadly primate feces, we determined recovery for each primate taxa with use the term “subadult” for all males aged between juveniles and adults each method. For the yellow baboons, 10,000 cpm of 125I testosterone (i.e., ages 4.5–8.0 years). We recognize that the subadult biological was added to 0.2 g of dry feces (Lynch et al., 2003). After incubation category is different for each of these taxa. for 1 h at room temperature, we proceeded with methanol extraction The ages of most yellow and chacma males were known to within and solid-phase extraction as described above. For the chacma a few days (for males born into study groups) and estimated based on baboons and geladas, we thoroughly mixed a large mass of feces in physical characteristics and date of immigration (for immigrating a plastic cup and measured out 10 aliquots of 0.5 ml wet feces. We males). By contrast, the ages of all gelada males were estimated based then added 18,000 cpm 125I testosterone tracer to each aliquot. After on developmental stages distinguished by physical markers (see incubating aliquots for 1 h at room temperature, we placed aliquots in Supplementary material). Therefore, to facilitate the comparison of 3 ml MeOH/acetone solution (4:1) and extracted each using the same profiles across all three taxa, we placed yellow and chacma males into methods described for each taxon above. Following elution, we multi-year age categories corresponding to those for gelada males. We measured recovery of the radioactivity with a gamma counter. then calculated a mean T value for each category (with each individual Recovery results for each taxa are listed in Table 1. contributing only one value). Testosterone profiles thus comprise a Second, we validated the DSL antibody for each taxon. We ran combination of longitudinal and cross-sectional data. After comparing serial dilutions of baboon and gelada fecal extract pools to check for T profiles based on the multi-year categories for all three taxa, we then parallelism with the standard curve. We also determined mean assay compared T profiles using single-year categories for yellow and accuracy (observed/expected×100) for each taxon by spiking each chacma males (single-year categories were not available for geladas). standard with an aliquot from the respective fecal pool and running them as samples. We calculated intra- and inter-assay coefficients of Maturational milestones variation for the assay for all three taxa. Results for parallelism, accuracy, and precision for each taxon are listed in Table 1. Male maturational milestones examined in this study include (1) Third, as a physiological validation to our methods, we compared T age at testicular enlargement (signaling puberty and the onset of concentrations between juvenile and adult males for all three taxa. subadulthood), (2) age at natal dispersal (when males leave their natal Although we did not expect age-related changes to be identical for all group and seek entry in another group), (3) age at attainment of adult three taxa, we did expect a consistent difference in which juvenile dominance rank (signaling the beginning of adulthood), (4) age at first males of each taxon would exhibit lower concentrations of T sexual consortship (the best measure available for age at first repro- metabolites than the fully adult males. duction in male primates), and (5) age at attainment of highest dominance rank (presumably the time of maximum mating opportunities). Age categories Milestones are based on individual life-history data for all except acquisition of highest rank, which, of necessity, is based on cross- Generally, primate juveniles are defined by maturational markers sectional data. We report median values for all five of these milestones such as independence from their mother (marking the start of juve- for yellow males (Alberts and Altmann, 1995a,b; Charpentier et al., nility) and puberty (marking the end). Because these maturational 2008; current study) and all but age of testicular enlargement for

Table 1 Validation results for yellow baboons, chacma baboons, and geladas.

Yellow baboons N Chacma baboons N Geladas N All assays N

Recovery (125I Testosterone %) 76.6a 5 65.1 10 55.9 10

Parallelism R2 0.97 14 0.99 12 0.98 24 Equation for observed against y=1.14 x−0.37 y=0.81x+6.12 y=0.88x+20.32 expected p b0.001 b0.001 b0.001

Accuracy R2 0.98 24 0.99 24 0.98 24 Equation for observed against y=1.14x−2.94 y=0.99x−3.06 y=0.99x+8.30 expected p b0.001 b0.001 b0.001

Precision Inter-assay CV (%) High kit control 11.70 66 Low kit control 14.09 66 Fecal pool (∼70 pg) 11.66 17 6.08 35 10.79 7 Intra-assay CV (%) High kit control 7.83 19 Low kit control 8.17 18 Fecal pool (∼70 pg) 6.27 12 4.76 10 4.29 10 a Lynch et al. 2003.

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Table 2 males had intermediate levels (Z-scores near zero, with the exception Median age (or age range, in years) that males reach hormonal and life-history of yellows, see below). milestones for yellow baboons, chacma baboons, and geladas.

Yellow N Geladas N Chacmas N Three-taxa testosterone comparison Hormones (testosterone production) Onset of adult T levelsc 6.5–7.5 — 6.5–8.0 — 7.5–8.5 — Consistent with the physiological validation, T trajectories were Peak T levelsc 7.5–8.5 — 6.5–8.0 — 8.5–9.5 — broadly similar across age for all three taxa. Nonetheless, two notable Life history (maturational milestone) differences were observed. First, yellow and gelada males attained a ———— Testicular enlargement 5.38 96 – First dispersal 7.47a 93 4.5–6.5b 70 9.252d 26 adult T levels at an earlier age than chacma males did (Figs. 1a c). Acquisition of adult rank 7.38a 48 N/A — 8.67d 15 Testosterone Z-scores for both yellow and gelada males changed from First sexual consortship 7.87a 31 6.5–8.0e 13 8.44d 6 negative to positive between 6.5 and 8.0 years of age, whereas those c Acquisition of highest rank 8.5–9.5 — N/A — 9.5–10.5 — for chacma males did not do so until 8.0–9.5 years. The ANOVA analysis a Source: Charpentier et al. 2008, known ages. of log T across age categories further supports this observation b Source: Dunbar and Dunbar, 1975 (p. 59), estimated ages. (ANOVA: yellows F(7,230)=4.85, p b0.001; geladas F(7,100)= c Based on cross-sectional data (see Fig. 1 for sample sizes for each age category). 11.13, pb0.001; chacmas F(6,50)=2.98, pb0.05; for post hoc tests N/A, not applicable. – d Source: Current data set, estimated ages. between successive age categories, see Figs. 1a c). None of the juvenile e Source: Current data set, known ages. chacma age categories by themselves was signiﬁcantly different from

chacma males (Table 2). For geladas, we report age ranges for dispersal and ﬁrst sexual consortship. Age at testicular enlargement is not known for either gelada or chacma males, and dominance ranks are not available for gelada males because, to our current knowledge, gelada males do not have a formalized dominance hierarchy.

Data analysis

As expected, none of the hormone values for the three taxa were normally distributed. Therefore, we log-transformed values prior to all analyses to facilitate the use of parametric statistics. Additionally, to avoid bias from uneven fecal sample distribution across individuals, we calculated a mean hormone value for each male for each age category and used this value in our analyses. All analyses were conducted separately for each taxon. Statistical analyses were performed using SPSS (16.0), and the statistical threshold for all analyses was set at pb0.05. First, for the physiological validation, we compared the T levels of juveniles to adults using a Student's t-test. Second, because T extraction efﬁciencies were different across the different methods for the three taxa, we standardized the three data sets using Z-scores to facilitate hormone comparisons across taxa. Z-scores represent the number of standard deviations that T values for each category (in this case, age groups) differ from the mean T value for that taxon (with negative Z-scores indicating values lower than the mean, and positive Z-scores indicating values higher than the mean). Z-scores were calculated for all three taxa across the multi-year age categories (see Supplementary material) as well as the single-year categories for yellows and chacmas. Third, we used one-way ANOVA on the log-transformed T values (not Z-scores) to compare hormone values across multi-year age categories. Because sample sizes were low for chacma juveniles, we increased statistical power by combining juveniles and subadults aged 4.5– 6.5 years into one category (chacmas only).

Results

Physiological validation

Juvenile males (2.5–4.5 years old) had signiﬁcantly lower T metabolites than adult males (N8.0 years old) for all three taxa (t- test: yellows, t(111)=−4.20, pb0.001; chacmas, t(39)=−2.17, Fig. 1. Testosterone Z-scores for the different age categories of (a) yellow males, (b) pb0.05; geladas, t(68)=−5.90, pb0.001). Additionally, T metabo- gelada males, and (c) chacma males. Although the ﬁgure depicts testosterone Z-scores, lites in all three taxa exhibited an inverse U-shaped pattern across age all statistical analyses were conducted on the log-transformed testosterone values. Due to the small sample size of juveniles for chacma age categories, we pooled juveniles and categories (Figs. 1a–c). In general, juveniles and subadults had the subadults age 4.5–6.5 years into a single age category for statistical testing. Tukey's post lowest T metabolites (negative Z-scores), adult males had the highest hoc tests: ⁎pb0.05, ⁎⁎pb0.01, ⁎⁎⁎ pb0.001. The number of males in each age category is levels of T metabolites (positive Z-scores), and the oldest of the adult indicated above the y-axis.

6 J.C. Beehner et al. / Hormones and Behavior xxx (2009) xxx–xxx the 8.0- to 9.5-year age category; however, this was likely due to males for both hormonal and maturational markers (Figs. 2a and b). insufficient power with this data set (i.e. sample sizes were low for Males of both taxa (1) exhibited a significant rise in T (Z-scores chacma males b6.5 years). When we pooled chacma juvenile males changing from negative to positive), (2) followed 1 year later by and males from the 4.5- to 6.5-year category, we detected a signi- lifetime maximum T, which coincided with attainment of adult rank, ficant difference from the 8.0- to 9.5-year age category for chacma (3) followed approximately 1 year later by a decrease in T levels (by at baboons. least −0.5 SD), which coincided with males' highest dominance rank. Second, the duration of elevated T profiles was shorter for gelada The delayed rise in T for chacma males coincided with delays in all and chacma males than for yellow males. Testosterone Z-scores for maturational milestones (dispersal, adult rank, and first sexual gelada males dropped quickly after 9.5 years (to nearly zero) and consortship) as compared to yellow males (or, where relevant, gelada scores for chacma males were negative after 11.0 years (Figs. 1b and males; Table 2). Chacma males also exhibited a drop in T levels after c). In contrast, Z-scores for yellow males, while dropping slightly 11 years (as indicated by negative Z-scores), while yellow males after 9.5 years, remain positive even in males 13 years and older (Fig. continued to exhibit higher than average T levels until 17 years of age 1a). Once again, post hoc tests from the ANOVA support these (as indicated by positive Z-scores). observations (for post hoc tests between successive age categories, see Figs. 1a–c). Gelada males exhibited significantly lower T levels by Discussion 9.5 years, and although chacma males did not exhibit significantly lower T levels until 13.0 years, Z-scores were negative by 11.0 years. Despite broad similarity in testosterone profiles across age groups By contrast, yellow males maintained high T metabolite levels and of all three taxa, we observed variability across taxa in the timing of did not exhibit a significant drop between any pair of successive adult two major testosterone transitions. One of these transitions was the age categories. attainment of adult levels of testosterone. While yellow and gelada Peak T levels for all three species coincided most closely with age males exhibited maximum testosterone between 6.5 and 8.0 years of at first sexual consortship (although resolution in geladas was coarse age, chacma males did not reach maximum testosterone until 8.0– due to our estimated age categories). For both yellow and chacma 9.5 years of age. Yearly testosterone profiles indicated that the chacma males, acquisition of adult rank came after the onset of adult T levels delay in testosterone production was 1 year later than that for and before the onset of peak T levels. Age of highest average rank yellows. The other major transition was the drop in testosterone that followed peak T levels by 1 year. males exhibited after an approximately 3-year period of maximal production. This drop was significant for geladas and chacmas but not Yearly testosterone comparison: yellows and chacmas for yellows. Although the testosterone of yellow males decreased gradually, levels did not fall to a consistently lower level until males Yellow and chacma males followed a similar pattern of maturation, reached 18 years of age. Consequently, for geladas and chacmas, the except that chacma males were approximately 1 year behind yellow period of maximal testosterone production was discrete (i.e., higher than all other age categories by at least one standard deviation) while for yellow males, testosterone production gradually tapered off as males senesced. To understand these differences, we examined testosterone profiles for each taxon in relation to several maturational milestones. We recall at this point that our main objective in this study was to describe taxon-level patterns in testosterone profiles across the male life span. We do not yet have available longitudinal, individual- based data across ages to partition variability into within and between taxa sources of variance, nor can we yet evaluate hormonal responses to specific social situations for the males involved. Rather, as a first step toward comparative endocrinology for wild populations, we take a broad look at overall hormone patterns and how male milestones map onto these profiles. Specific investiga- tions into the relationship between testosterone changes and male developmental markers for individual males are important topics for the future. As such, the present analysis offers a framework for formulating and testing specific hypotheses for such subsequent studies.

Testosterone and maturational milestones

First, the three taxa exhibited considerable variability in the relationship between testosterone and natal dispersal. At one extreme, geladas dispersed well before the attainment of adult testosterone levels. Gelada “dispersal” from natal one-male units, however, may be qualitatively different from dispersal from natal groups in baboons because the first of these dispersal events in geladas occurs during the subadult (and even juvenile) stages (Dunbar and Dunbar, 1975). Juvenile and subadult gelada males repeatedly come and go from all-male bachelor groups, returning to their natal one-male unit each time, making it difficult to establish a Fig. 2. Testosterone Z-scores (grey bars) and ordinal dominance ranks (±SEM, black final natal dispersal event. At the other extreme, chacmas dispersed dots) across 1-year age categories for (a) yellow and (b) chacma males. The number of males in each age category is indicated above the y-axis. Male maturational milestones about a year after attainment of adult testosterone levels. Natal (see text) are indicated by arrows. dispersal for chacma males is delayed not only with respect to

J.C. Beehner et al. / Hormones and Behavior xxx (2009) xxx–xxx 7 testosterone but also relative to all male milestones for the other two may also have delayed Moremi chacma males' life-history variables taxa. A model of the ecological effects on dispersal (Alberts and since dispersing males are certain to experience elevated mortality Altmann, 1995a) considers the trade-offs that dispersing males of all during transfer. Thus, pressures related to ecological factors in this ages face between opportunities for mating (which increase with area may have selected for a reproductive strategy that maximizes population density) and possibility of mortality (which increases “maturation” in one's natal group prior to dispersal. Chacma males with predation rate). However, the model does not consider the dispersed and attained their highest dominance rank in the same year. temporal aspect of natal dispersal within the life-history trajectory of This “strategy” is in sharp contrast to yellow males, and possibly even a taxon. Indeed, for both the yellow and chacma populations studied anubis (P. anubis or P. h. anubis) males; both yellow and anubis males here, many males remain to breed for some time in their natal group disperse around 8 years of age (Charpentier et al., 2008; Packer, 1979; (Alberts and Altmann, 1995a; Bulger and Hamilton, 1988), possibly Packer et al., 1995), yet do not attain highest rank until nearly 2 years because (as per the model) Amboseli yellow baboons live at later for yellows (Table 2) and 3 years later for anubis (Packer et al., relatively low densities (Altmann and Alberts, 2003), and Moremi 2000). chacma baboons experience relatively high predation (Cheney et al., At the other end of the life-history trajectory, why do yellow 2004). Perhaps as a consequence of multiple ecological factors males continue to produce testosterone well beyond the period impinging on dispersal timing, we found no clear relationship when they are high ranking? Low-ranking male yellow baboons have between the attainment of adult levels of testosterone and dispersal comparatively greater access to sexually receptive females than male for these taxa. geladas and chacmas (Alberts et al., 2003, 2006). Two components of Second, the age at first sexual consortship was approximately the this weak, dominance-based priority are that younger, non-alpha same time as that of peak testosterone production. However, under males are able to gain some fertile matings and that older, non-alpha the predictions of the challenge hypothesis, sexual activity alone males are also able to mate more than would be expected under a should not stimulate an exponential increase in testosterone strict queuing scenario. This prolonged maintenance of higher production (Wingfield et al., 1990) but rather sexual activity in testosterone levels into older ages for yellow males represents a concert with male–male aggression, that, for baboons, occurs in the departure, not only from chacma and gelada males but also from context of rank attainment within a dominance hierarchy. Because the other known mammalian male profiles (Castracane et al., 1986; age of first sexual consortship and the acquisition of adult rank were Crawford et al., 1997; Muehlenbein et al., 2001; Seraphin et al., temporally similar for yellows and chacmas (b5 months apart for 2008). yellows and b3 months apart for chacmas), the present analysis is not fine tuned enough to sufficiently relate peak testosterone levels to Testosterone and parental care either of these milestones. Third, yellow and chacma males attained their highest dominance In many ways, male development for chacma baboons is more rank 1 year after peak testosterone production. This supports an similar to that of geladas than it is to that of yellows. Compared to the earlier finding that baseline testosterone levels predict future testosterone profiles of yellow males, those of chacma and gelada dominance ranks, but rank changes in themselves do not affect males indicate a comparatively discrete increase to adult levels and testosterone levels (Beehner et al., 2006a). Our results support the decrease about 3 years later. This discrete period of elevated challenge hypothesis in showing a close link between testosterone testosterone production is what we might expect for a species such and a time period when we expect male–male contests, but the as the gelada, with unimale reproductive units. That chacmas also fit causality of this relationship (if any) is opposite the prediction. The this profile suggests that chacma male physiology may be tracking challenge hypothesis was proposed as a feedback loop between the reproductive activity and/or parenting behavior better than the external social environment and the internal physiological one, with a overall mating system. Although yellow and chacma baboons share a social “challenge” initiating the cycle. However, for both yellow and multimale, polygynandrous mating system, the prime “mating” chacma males, testosterone declines before males fall in rank— versus “parenting” stages for chacmas and geladas may be more sometimes even up to 6 months beforehand (Beehner et al., 2006a). compartmentalized than those of yellows. Although older, low- Unless there is an anticipatory decline in testosterone, our data ranking chacma males continue to take advantage of mating indicate that testosterone production is not necessarily “socially opportunities (Crockford et al., 2007), and they achieve some modulated” (Wingfield et al., 1990)—or, at least not at the broad scale reproductive success (Cheney and Seyfarth, unpublished data) they that we use in this study. Individual contests may indeed affect daily also invest considerable time and energy in paternal care (Moscovice fluctuations in testosterone levels (i.e., winner-loser effects; Mazur et al., 2009; Palombit et al., 2000, 1997). In many mammalian species, and Booth, 1998; Mazur et al., 1992). However, we suggest that the high levels of testosterone have been shown to be incompatible with more stable baseline testosterone levels that characterize life-history paternal behavior (Gray et al., 2006; Muller et al., 2009; Nunes et al., stages do not result from rank changes but, in fact, precede them. 2000, 2001), and thus the chacma male sharp decline may be related to a shift in from an overall mating strategy to a parenting one. Testosterone differences across taxa Taxon or population differences? Why do chacma males delay testosterone production, rank acquisition, and dispersal by at least a full year relative to yellow One factor that has not been addressed in this study is whether the males? One explanation may relate to the high density of baboons in male hormone and life-history patterns we report here are taxon the Moremi chacma population (24 baboons/km2; Cheney, 1987; specific or population specific. This raises the question of whether we Cheney et al., 2004; Hamilton and Bulger, 1992; Hamilton et al., 1976). are documenting intrinsic or extrinsic sources of variation. The overall Males attempting to disperse to neighboring groups may face high hormonal profile may exhibit a fixed pattern within a taxon and tell us resistance from the males already established in these groups. Males something about a taxon's overall adaptive strategy. Alternatively, might overcome this resistance by achieving full adult body size prior different populations may demonstrate facultative responses to to emigration since increased body size upon immigration to a new environmental changes, and hormones (such as testosterone) can group could facilitate a more rapid ascent in the dominance hierarchy. facilitate this behavioral flexibility (Oliveira, 2004). Offering some Additionally, if males rise in dominance within their natal group, they support to the latter hypothesis, captive yellow or yellow-olive hybrid may gain more “practice” at rank contests, whether this involves males exhibited adult levels of testosterone much earlier (Altmann et actual fighting or displaying (Kitchen et al., 2003). High predation al., 1988; Castracane et al., 1986; Muehlenbein et al., 2001) than the

8 J.C. Beehner et al. / Hormones and Behavior xxx (2009) xxx–xxx wild yellow males in the current study. At present, no comparable Animals at the University of Michigan (UCUCA protocol #09554) and hormone data are available from other wild populations of baboons or adhered to all the laws and guidelines of Ethiopia. geladas to test these alternatives. Certainly, a replication of this study Finally, we would like to thank T. Bergman and two anonymous in other populations with different population dynamics and/or reviewers for their helpful comments on earlier versions of this ecology would help resolve this issue (for details on the ecology of manuscript. these three sites, see Amboseli (Alberts et al., 2005; Behrensmeyer, 2006), Moremi (Ellery et al., 1993), and Simien (Nievergelt et al., Appendix A. Supplementary data 1998)). For example, if the delay in testosterone production and other life-history stages observed in chacma baboons is due to the high Supplementary data associated with this article can be found, in population density in Moremi, then we predict a very different the online version, at doi:10.1016/j.yhbeh.2009.08.005. pattern for chacma baboons at lower densities (e.g., Drakensberg chacma population, South Africa). A third possibility (which is obscured using the broad-scale References approach we use here) is that testosterone variation represents en- Alberts, S.C., Altmann, J., 1995a. Balancing costs and opportunities: dispersal in male during individual variation and thus reflects alternative life-history baboons. Am. Nat. 145, 279–306. strategies. For example, a high parental investment strategy may Alberts, S.C., Altmann, J., 1995b. Preparation and activation: determinants of age at reproductive maturity in male baboons. Behav. Ecol. Sociobiol. 36, 397–406. exhibit a long-term commitment to one mate and parental care, Alberts, S.C., Altmann, J., Wilson, M.L., 1996. Mate guarding constrains foraging activity accompanied by low mating effort, while a low parental investment of male baboons. Anim. Behav. 51, 1269–1277. strategy may exhibit a low commitment to one mate and no paternal Alberts, S., Watts, H., Altmann, J., 2003. Queuing and queue-jumping: long-term patterns of reproductive skew in male savannah baboons, Papio cynocephalus. care, accompanied by high mating effort (Gross, 1985, 1996; Anim. Behav. 65, 821–840. Thompson and Moore, 1992; van Rhijn, 1974). With respect to this Alberts, S.C., Hollister-Smith, S.C., Mututua, R.S., Sayialel, S.N., Muruthi, P.M., Warurete, third possibility, future studies that statistically examine the variabil- J.K., Altmann, J., 2005. Seasonality and long term change in a savannah fi environment. In: Brockman, D.K., van Schaik, C.P. 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Please cite this article as: Beehner, J.C., et al., Testosterone related to age and life-history stages in male baboons and geladas, Horm. Behav. (2009), doi:10.1016/j.yhbeh.2009.08.005